The present invention relates in general to methods of determining or monitoring the weight of organic molecules. It relates in particular to time of flight (TOF) measurement methods using laser desorption ionization of molecules to be measured.
Time of flight methods of determining molecular weight are normally used for large molecules such as organic molecules (having a molecular weight greater than about 100,000 daltons). Such molecules are generally so heavy that many well known mass spectrometric methods, such as magnetic sector and quadropole magnetic sector are ineffective. For magnetic sector mass spectrometry, ionized species are produced in a vacuum and passed through a magnetic field. The magnetic field is varied in intensity to selectively guide analyte towards a detector. A correlation between magnetic intensity and molecular charge ratio is used to determine analyte mass. Large organic molecules may be sufficiently heavy in relationship to their charge (charge/mass ratio) that they are not readily deflected by such magnetic fields. In an effort to compensate for this shortcoming, some techniques, such as electrospray, rely upon the generation of high multiple charges (10-50) to facilitate the analysis of large molecular weight constituents. However, such approaches yield multiple signals for a single analyte, thus greatly increasing the complexity of data interpretation.
In time of flight methods of mass spectrometry, charged (ionized) molecules are produced in a vacuum and accelerated by an electric field produced by an ion-optic assembly into a free flight tube or drift tube. The velocity to which the molecules may be accelerated is proportional to the square root of the accelerating potential, the square root of the charge of the molecule, and inversely proportional to the square root of the mass of the molecule. The charged molecules travel, i.e. "drift" down the TOF tube to a detector. Since the total travel distance for molecules is fixed and since velocity is defined as distance traveled divided by travel time, it becomes apparent that the molecular weight of a molecule is proportional to the square of its flight or travel time.
Laser desorption ionization mass monitoring is a TOF mass monitoring method wherein charged molecules of a species to be measured, or analyzed, are produced by laser irradiation (in vacuum) of a crystalline host matrix including a small proportion, for example between about 1:1000 and 1:100,000 of the species. The wavelength of the incident radiation is dependent upon the absorbance characteristics of the matrix. Thus, the host matrix is selected to optimally absorb the radiation. The absorption of this energy results in the ejection or desorption of analyte molecules from the matrix in the form of charged molecules (ions). The desorbed, charged molecules are then accelerated into a drift tube by electric fields produced by an ion optic assembly. The time of flight of the molecules through the tube is generally determined by detecting the irradiating pulse and using the detected signal to start a timing process. Charged molecules generated by the irradiating pulse are intercepted by a detector after they have traversed the drift tube. The time interval existing from the start signal to the detector pulse establishes the time of flight. Molecular weight to charge ratio of the analyte is determined using the following function: EQU M/Z=F.multidot..tau..sup.2 +C
where .tau. is the flight time, F is a factor dependent upon flight distance and molecular kinetic energy, and C is a constant. The values of F+C are determined by calibrating the function using a standard of predetermined molecular weight.
Two classifications of LDIM instruments have been established, microprobe instruments and bulk analysis instruments.
In a microprobe instrument, laser irradiation is finely focused to a small spot on a foil containing the analyte. The laser radiation is in the form of a short pulse of very high power density. The power density is such that a small hole is produced in the foil. Analyte ions are desorbed from the foil and emerge from the hole. A commercially available LDIM microprobe instrument is described in Hillenkamp, F., et al., Int. J. Mass Spec. Ion Physics, vol. 47, 1983, 19-22.
Bulk analysis instruments use moderately focussed beams, for example, beams focussed to a spot having an area greater than about 0.1 square millimeters. The beams are incident on a surface including the analyte in a host matrix. The matrix and analyte are applied in the form of a thin crystallized layer or layers on a surface forming the tip of a sample probe. In the bulk analysis instrument, an area on the probe tip may be irradiated sequentially, with multiple laser pulses. This may be helpful, for example, in gathering statistical data on measurements. Additionally, the irradiated region of the probe may be off its central axis, allowing the desorption of several different probe regions to be achieved by rotating the probe tip. Alternatively, such a probe may be sequestered into several different circumferential sample regions in which the off axis array permits the simultaneous introduction of multiple samples into the ion source.
In prior art ionization methods used in mass spectrometry, energetic or "hard" ionization processes, for example, using energy exchange within a gas discharge, may produce fragmentation of analyte molecules, i.e. the formation of metastable ions having a range of different weights.
In both microprobe and bulk LDIM methods the laser desorption ionization method produces what may be termed "soft ionization" of an analyte. Soft ionization provides that predominantly single charged unfragmented analyte ions are generated. Preferably, ions are desorbed by a laser pulse having an intensity just above that threshold intensity required to cause desorption. In a pulsed laser it is difficult to provide pulses of repeatable intensity particularly if a laser is operated intermittently. Further, thresholds may vary between matrix sample combinations. If a pulse has an intensity significantly greater than the desorption threshold, adducts may be formed by the addition of one or more matrix molecules to a sample. This causes a distribution of indicated molecular weights around a true value leading to measurement uncertainty or loss of mass resolution. Additionally, variance in laser power can cause variability in the total kinetic energy of analyte molecules. Such variances create a reduction in resolution when successive scans for a given sample probe are summed.
In LDIM, resolution is determined by M/.DELTA.M where M is the assigned mass value for the analyte (usually the peak centroid) and .DELTA.M is the molecular weight width of the peak at its half height (this corresponds to the molecular weight distribution for the peak at its first moment). This is a measurement of an instrument's ability to produce separate signals from ions (molecules) of similar mass. LDIM mass resolution is dependent upon the molecular weight of an analyte. Generally, mass resolution decreases as analyte molecular weight increases. The latter may be attributed to increased molecular population heterogeneity of composition and initial desorption velocity distribution.
Instrumental limitations to resolution primarily arise from uncertainties in flight time measurement, variance in flight path distance, and variance of total molecular kinetic energy.
Uncertainty in flight time measurement stems from the error in determining flight time duration from one desorption event to the other. Such error originates in timing jitter inherent to electronic trigger and detection circuitry. When successive desorptive events (sample scans) are summed in an effort to increase signal to noise performance, this jitter phenomenon creates a broadening of the summed signal.
In order for the time-of-flight experiment to work, the total flight distance and total kinetic energy for a given population of molecules must be constant. If all molecules of a given population do not travel along the same flight path, a variance of flight distance will result. This variance will create a distribution of flight times for identical molecules, resulting in an increase in the molecular weight distribution for that population at its first moment. The result is a broadening of the signal created by each individual scan. Alterations in flight path may be due to discontinuities of ion optic electrical fields, molecular collision during free-flight, and thermal-induced axial diffusion.
The total kinetic energy imparted to charged particles in the time-of-flight experiment is the sum of the initial kinetic energy transferred to the analyte by the laser pulse and the kinetic energy transferred in the ion optic, acceleration region. Alterations in laser pulse intensities from one desorptive event to the other create a variance of total kinetic energy for this series of events. This variance, in turn, results in the broadening of the resultant signal when such events are summed, reducing resultant resolution.
In a similar manner, ion-optic, acceleration electrical field variances cause alterations in molecular total kinetic energy. These variances may be due to electrical instability of the ion components (coronal discharge, current leakage, current limitations, etc.) or alterations in DC voltage intensity due to the presence of AC ripple.
A preferred detector for LDIM instruments is a microchannel plate MCP detector. Such a detector consists of one or two microchannel plates. Two plates are arranged in a tandem array. Each plate consists of a plurality of microscopic tubes which are held in an electric field. Ion collisions with the wall of these tubes incite the release of electrons. These electrons then cascade down these tubes releasing more electrons. This results in a conversion of electrical charge from ions to electrons with a simultaneous increase in total charge. These electrons are then utilized by electronic circuitry to produce a signal.
Ion detection sensitivity may be enhanced by employing a mass filter. A mass filter is a pulsed, electrical field applied perpendicular with respect to ion propagation just after the ion acceleration region. Mass filtering is achieved when this perpendicular electrical field deflects ions from the proper propagational path during its energized state. Consequently, these deflected ions never strike the detector, thus preserving the available electrons required for the microchannel plate cascade phenomenon. Such an arrangement may be used as a high-pass mass filter if careful and accurate timing algorithms are employed to deflect analyte of the desired mass by applying the deflection pulse only during the time period in which the unwanted analyte is resident in the deflection field. The field must then rapidly collapse to allow subsequent ions to pass through into the remainder of the free-flight zone, unaltered in their trajectories.
Another source of uncertainty in LDIM measurements is the formation of ions of the same molecule having different charges or clusters of two or more molecules having one or more unit charges molecules. These may be referred to as quasi molecular ions and will have different flight times in an LDIM instrument. As such, they may indicate that different molecules are present in a sample and thus lead to difficulty in assessing the purity of a sample.
Still another source of uncertainty in LDIM measurements may lie in the preparation of samples. It is important to lay down an even, reproducible co-crystalline layer of matrix and analyte on a sample probe tip. In order to insure regular non-biased co-crystallization of matrix and analyte, the crystallization event must occur rapidly, on the order of five to fifteen seconds. Typically, a sample-matrix mixture consists of organic and aqueous solvent components. Such a solvent mixture is required to solvate matrix and analyte molecules, possessing both hydrophobic and hydrophilic characteristics. If co-crystallization slowly progresses, solvent composition gradually become increasingly hydrophilic as the more volatile, organic constituents vaporize. Accordingly, we see a temporal based crystallization order in which hydrophobic solutes crystalize first followed by hydrophilic solutes. For the most part, matrix molecules are largely hydrophobic. Consequently, matrix molecules will preferentially co-crystalize with sample solutes of low hydrophilicity, biasing the resultant ion signal against hydrophilic sample components. Rapid co-crystallization avoids these problems. A preferred means of crystallization is through the use of vacuum application. A droplet of homogenous matrix/analyte solution may be applied to a probe tip. The drop is then crystallized by applying a vacuum to the probe tip to remove volatile fluid components. If the droplet is irregular in shape then thickness and sample distribution in the crystallized layer can be nonhomogeneous leading to unreproducible measurement results. If vacuum application is not variable, highly lipophilic analyte/matrix mixtures will be difficult to crystalize since more volatile components of the mixture will cause less volatile components to bubble.
U.S. Pat. No. 5,045,694 discloses an electrospray method of applying matrix to a probe tip. Although this method appears to produce better matrix layers, it involves applying a potential of about five thousand volts to the probe tip during application of the layers. This makes the method somewhat hazardous and can lead to corona discharge between the probe tip and the spray apparatus which may damage the probe tip and spray apparatus.
In view of the foregoing it will be evident that although LDIM provide a potentially convenient method for monitoring molecular weight of large organic molecules, there is a need for improvement in many hardware aspects of the technology including sample preparation, delivery of laser and deflection pulses, ion optics, and detectors. There is also a need for improved signal processing technology to identify and eliminate uncertainties which may arise, particularly from the generation of quasi molecular ions.